Extracts of passion fruit and uses thereof

ABSTRACT

An extract of passion fruit is prepared. The extract has the effect of lowering blood pressure and serum nitric oxide levels in mammals. The extract also provides a hepatoprotective effect, as well as antioxidant and anti-inflammatory effects in mammals. A novel compound was identified in the extract and given the name edulilic acid.

RELATED APPLICATIONS

This application claims priority to New Zealand Patent ApplicationSerial No. 532138, filed Apr. 2, 2004.

BACKGROUND OF THE INVENTION

The invention relates generally to botanical extracts and, morespecifically, to extracts of passion fruit (Passiflora sp.), includingparticularly extracts of the skin of passion fruit, and the use of theextracts for food, nutraceutical, and medical applications.

Hypertension, or a blood pressure higher than 140/90 mm Hg, is the mostcommon risk factor for cardiovascular and cerebrovascular morbidity andmortality. In the United States, high blood pressure is responsible for40,000 deaths annually, while being the most modifiable risk factor forstroke. Hypertension affects about one in four adults, or almost 50million people in the United States.

A Framingham study showed that as people aged from 30 to 65 years, theirblood pressure increased an average 20 mm Hg systolic and 10 mm Hgdiastolic pressure, with systolic blood pressure continuing to rise upto age 90.

While higher blood pressure increases the likelihood of a cardiovascularevent, hypertension is not often well controlled and too few patientsare adequately treated. Epidemiologic studies predict that reduction ofthe systemic blood pressure by the amount usually achieved in majorclinical trials could reduce cerebrovascular events by 42% and cardiacevents by 24%

Hypertension is frequently treated non-specifically, resulting in alarge number of minor side-effects, and a relatively high rate of non-or inadequate treatment. Thus, the search for new treatments forhypertension remains ongoing.

Therapies derived from natural products are well known. It has beenestablished that certain flavonoids have a beneficial effect onhypertension. For example, a bark extract from the French maritime pine(Pinus pinaster), which contains a mixture of flavonoids, decreasessystolic blood pressure when taken orally by mildly hypertensivepatients.

Nitric oxide is an important molecular regulator of blood pressure.Nitric oxide is a potent vasodilator. It inhibits platelet activation,limits leukocyte adhesion to the endothelium, and regulatesmyocardiocontractility. Synthesis of nitric oxide catalyzed by nitricoxide synthase (NOS) occurs in the vascular endothelium while theproduction of nitric oxide involving inducible nitric oxide synthase(iNOS) is associated with immune function. However, small amounts ofnitric oxide produced by another NOS, epithelial nitric oxide synthase(eNOS), have a cytoprotective effect and vasodilation action on thecardiovascular system.

Peroxynitrite is a potentially damaging oxidant, formed from nitricoxide (NO+O₂ ONOO⁻). Peroxynitrite can give rise to lipid peroxidation,protein nitration, DNA single-strand breakage, and guanidine nitration.

It has been shown that the flavonoids quercetin and kaempferol inhibitNOLA-dependent spontaneous aortic ring contraction in spontaneouslyhypertensive rat (SHR) cells in vitro. NOLA is a nitric oxide synthaseinhibitor. Large dose acetylcholine-induced vascular contraction canalso be inhibited by antioxidative flavonoids such as quercetin,kaempferol, rutin, and esculetin. Inhibition of vascular smooth musclecontraction should lead to lower blood pressure.

In addition, the effects of flavonoids on immune function arecontroversial. Catechin enhances proliferation of lymphocytes andantibody production, while it exerts an inhibitory effect at highconcentration. Some studies show that flavonoids enhance NK cellactivity, while other studies show that flavonoids have no effect.Quercetin seems to inhibit non-specific immunological responses andexerts an anti-inflammatory action.

Passion fruit (Passiflora edulis) is a subtropical or tropical plantwith a vigorous climbing character, growing to 20 ft. The purple passionfruit is native from southern Brazil through Paraguay to northernArgentina. Its fruit is nearly round or ovoid, 1.5 to 3 inches wide,with a tough, smooth and waxy rind.

In a search for bioactive constituents of passion fruit, it has nowsurprisingly been found that a passion fruit extract lowers systolicblood pressure in spontaneously hypertensive rats (SHR), and decreasesnitric oxide production from iNOS, thus improving endothelialdysfunction in SHR. It is therefore also envisaged that the passionfruit extract will exhibit antioxidant properties.

It is therefore an object of the invention to provide an extract ofpassion fruit which exhibits therapeutic effects against hypertension,and diseases associated with hypertension, and which ishepatoprotective, or at least to provide a useful choice.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a method of lowering bloodpressure in a mammal comprising administering an effective amount of apassion fruit extract to the mammal.

In another aspect, the invention provides a method of preventing ortreating a disease or disorder in a mammal where it is desirable tolower blood pressure comprising administering an effective amount of apassion fruit extract to the mammal.

There is also provided a method of lowering serum nitric oxide levels ina mammal comprising administering an effective amount of a passion fruitextract to the mammal.

The invention further provides a method of treating a disease ordisorder related to liver function in a mammal comprising administeringan effective amount of a passion fruit extract to the mammal.

The invention therefore provides a method of treating hypertension aswell as any other disease or disorder associated with elevated bloodpressure. The invention further provides-a method of hepatoprotection ina mammal, as well as a method of treating any disease or disorderrelated to liver function.

The invention further provides the use of a passion fruit extract as anantioxidant to inhibit damage from free radicals, to reduce serum lipidperoxidation and to preserve healthy tissue antioxidant vitamin levels.

The passion fruit extract of the invention includes one or more of thegroup selected from quercetin, cyanidin glycoside, catechin,epicatechin, luteolin, phenylpyruvic acid, the novel edulilic acidisolated and described herein, and any glycoside thereof.

Preferably, the passion fruit extract is prepared by a process includingthe following steps:

-   -   (i) preferably, cutting the passion fruit into pieces to        increase the surface area;    -   (ii) contacting the pieces of passion fruit with water to give        an aqueous extract and a solid residue;    -   (iii) separating the aqueous extract from the solid residue;    -   (iv) contacting the aqueous extract with a polymeric matrix to        adsorb one or more components of the extract onto the matrix;    -   (v) washing the matrix with water; and/    -   (vi) eluting the one or more components from the matrix with an        organic solvent or mixture of organic solvents.

Optionally, the skin of the passion fruit is removed from the flesh andused in the extraction process.

It is preferred that the organic solvent is methanol, ethanol, isopropylalcohol, 1-propanol, or acetone.

The invention also provides a composition containing the passion fruitextract.

The composition may be a food or food product. The composition may alsobe a dietary supplement, such as a nutraceutical or other nutritionalcomposition.

Alternatively, the composition may be a pharmaceutical compositioncomprising the extract described above, admixed with one or morepharmaceutically acceptable excipients.

In a further aspect, the invention provides the use of a passion fruitskin extract as a nutraceutical, such-as a dietary supplement, or as anactive ingredient in the preparation of medical or functional foods andbeverages.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1 a-c are HPLC traces of the extract of passion fruit madeaccording to the present invention.

FIG. 2 is a diagrammatical representation of data showing the reductionin blood pressure in a group of spontaneously hypertensive ratsadministered the extract of passion fruit.

FIG. 3 is a diagrammatical representation of data showing the reductionin serum nitric oxide levels in a group of rats administered the extractof passion fruit.

FIGS. 4 a and 4 b are diagrammatical representations of data showing thein vivo increase in activity of Na—K ATPase and Ca ATPase, respectively,by the extract of passion fruit.

FIGS. 5 a and 5 b are diagrammatical representation of data showing thereduction in systolic blood pressure and diastolic blood pressure,respectively, in a group of humans administered the extract of passionfruit.

FIGS. 6 a and 6 b are drawings showing H-1 methylene proton interactionswith H-1′ and H-2′ of the sugar moiety.

FIGS. 7 a and 7 b are HPLC-ELSD chromatograms of the extracts of passionfruit skin and pulp, respectively, obtained under identical conditions

DETAILED DESCRIPTION

As described herein, “passion fruit” means generally the fruit includingboth the skin and the edible pulp. The term “passion fruit skin” is usedto mean the remaining part of the fruit after the edible pulp inside hasbeen removed. HPLC analysis indicates that the passion fruit extract ofthe invention contains a number of flavonoids, including quercetin,quercetin galactoside, quercetin glucoside, luteolin, luteolinglucoside, cyanidin-3-glucosides, catechin and epicatechin. (FIG. 1).

The flavonoid and cyanidin components of the extract inhibit superoxideformation and nitric oxide production from iNOS, thus improvingendothelial dysfunction and lowering blood pressure. Quercetin has beenshown to inhibit iNOS mRNA and the production of nitric oxide.

Human essential hypertension is characterized by impairedendothelium-dependent vasodilation, caused by oxidative stress. Theextract of the invention has anti-hypertensive effects. In addition, theflavonoid components of the extract lower blood pressure, inhibitoxidation of LDL, and inhibit platelet aggregation, thereby exerting acardiovascular protecting action. It is noted that the components of theextract include quercetin, a compound know to have antihypertensiveactivity, but the amount contained in the extract is less than betweenabout 1 and 5% and is not sufficient to substantially account for theantihypertensive activities of the extract. Indeed, none of thepreviously known constituents of the extract are present in quantitiessufficient to provide the observed effects alone.

It is also envisaged, based on the in vitro data, that the extract willhave antioxidant properties.

In studies with spontaneously hypertensive rats (SHR), the applicantsfound that diets supplemented with 50 mg/kg of the extract lowered bloodpressure in SHR, retarding their normal increase in blood pressure dueto aging. Systolic blood pressure was 12.3 mm Hg lower in rats fed 50mg/kg of the extract, compared with a control group (P<0.01) (FIG. 2).

Furthermore, nitric oxide concentration was 18.82 μmol/L in rats fedwith 10 mg/kg of the extract, 40% lower than that in rats fed noextract. The nitric oxide concentration was 11.07 μmol/L in rats fedwith 50 mg/kg of the extract, 65% lower than that in rats fed no extract(FIG. 3). This will prevent the overproduction of nitric oxide and itssubsequent reaction to form peroxynitrite which is detrimental to thecardiovascular system.

The applicants have also found, in rat liver studies, that the passionfruit extract is hepatoprotective. Precision-cut rat liver slices wereincubated with 20 μg/ml of passion fruit extract. At 9 and 24 hoursincubation, potassium levels in the slices, an index of viability, werenot significantly different from control slices.

The extract was then incubated with precision-cut rat liver slices inthe presence or absence of 1 mM chloroform, a hepatotoxicant. At 6 hoursincubation and at 9 hours incubation the passion fruit extract showedsignificant hepatoprotection against chloroform injury. No toxicity ofthe extract was found in this study.

In vitro studies have also shown that the extract increases human redblood cell membrane-bound Na—K ATPase and Ca ATPase activity. RBCmembrane-bound Na—K pump ATPase had much higher activity when culturedwith the extract at either 0.25 mg/mL (increased by 102%), 1 mg/mL(increased by 107%), or 25 mg/mL (increased by 170%) than the controlgroup (FIG. 4). The extract at concentrations of 1 mg/mL and 2.5 mg/mLincreased membrane-bound Ca ATPase activity by 78% and 41% on average.

The applicants have also carried out human studies (FIG. 5). FIG. 5shows the change in SBP (systolic blood pressure) and DBP (diastolicblood pressure) in hypertensive patients who received placebo or passionfruit extract (2 mg/lb/day, maximum 400 mg/day) for four weeks. Passionfruit extract or placebo pills were given in a randomized, double-blind,parallel group fashion to these patients. They had an average systolicblood pressure of 176.60±4.90 mm Hg (mean±SEM). Passion fruit treatmentdecreased systolic blood pressure significantly (p<0.001) to 145.67±4.44mm Hg (mean±SEM) as compared to the placebo group. The data alsodemonstrated that passion fruit extract supplementation decreasesdiastolic blood pressure significantly (p<0.001) in hypertensivepatients with an average diastolic blood pressure of 103.27±2.30 mm Hg(mean±SEM), to 78.67±2.78 mm Hg (mean±SEM). No patient in the studyshowed electrocardiographic changes after four weeks of therapy.

Because of the above-described activities, it is also expected that thepassion fruit extract will benefit patients with inflammatory-relateddiseases, such as arthritis, asthma and allergies, as well as heartdisease and hypertension. In addition, even though very large intakes oramounts of the extract were used in the studies described in thisapplication, no toxicity of the extract was found in humans, rats, miceor cells in culture.

It will be appreciated by those skilled in the art that the extract maybe administered to a patient by a variety of routes, including oraladministration, or injection. The amount of extract to be administeredwill vary widely depending upon the patient and the nature and extent ofthe disorder to be treated. Typically, the extract is formulated as acomposition which may be administered intravenously or by oralingestion. The composition may be ingested or intravenously administeredin any dosage levels and dosage frequencies suitable for lowering bloodpressure and/or increasing immune function.

The composition of the invention may also be a food product, including,but not limited to, a nutritional supplement.

In the case of a pharmaceutical composition, the extract may beformulated into solid or liquid preparations, for example tablets,capsules, powders, solutions, suspensions and dispersions. Liquid formsinclude carriers such as water and ethanol, with or without other agentssuch as pharmaceutically acceptable surfactants or suspending agents.

The invention is further described with reference to the followingexamples. However, it is to be appreciated that the invention is notlimited to these examples.

EXAMPLES Example 1 Preparation of Passion Fruit Extract

Passion fruits (Passiflora edulis) were cut into halves and the juicypulp removed to give empty shells of passion fruit skin. The shells werechopped into small pieces less than 10 mm in length and placed in acontainer. Hot water (65-75° C.) was added to the container to immersethe chopped shells completely. The mixture was stirred occasionallyduring the first hour and then left to soak overnight. The mixture wasfiltered and the filtrate passed through a column of non-ionic polymericresin to absorb phenolic and other organic compounds. Distilled waterwas passed through the column to wash out sugars and other polarcomponents. The absorbed compounds were then eluted from the column withmethanol and the eluant concentrated under reduced pressure to give adark concentrate. The concentrate was freeze-dried to give the passionfruit extract as a dark red powder. While methanol was used as theeluting agent, ethanol, isopropyl alcohol, 1-propanol or acetone couldalso have been used.

Example 2 Determination of Components of Passion Fruit Extract

The components of the extract were determined by HPLC. Experiments werecarried out on a Hewlett Packard 1100 instrument equipped with a DADdetector and a LiChrospher 100 RP-18 (um) column (125×4) held at 30° C.The solvent program started from 3.6% B (2% HOAc in acetonitrile) insolvent A (2% HOAc in water) up to 12% B in 20 min, to 20% in 30 min,and to 50% B in 45 min. Flow rate was set at mL/min and compounds weremonitored by UV absorption set at 280 nm for phenolic acids, 350 nm forflavonoids and 520 nm for anthocyanins. The identity of the compoundswas confirmed by comparison of retention times and UV/visible spectrawith authentic materials.

Example 3 Red Blood Cell Membrane Preparation and ATPase Assay

Erythrocyte membranes were prepared as previously described (Farrance, ML., & Vincenzi, F F. (1977) Biochim Biophys Acta 471:49-58). Briefly,blood was taken from healthy humans. Red blood cells were washed withsaline and lysed in a hypotonic imidazole buffer (pH 7.4, 20 mM, Sigma)with EGTA (100 mM, Sigma) and PMSF (10 mM, Sigma). Membranes were washedwith imidazole buffer (20 mM) containing EGTA and PMSF, imidazole buffercontaining EGTA, and only imidazole buffer each one time in sequence.The final wash was in 40 mM histidine-imidazole buffer (pH 7.4), and themembranes were stored in a refrigerator (4-8° C.) under nitrogen. Priorto assay, RBC membrane (0.75 mg/mL) was incubated for 30 minutes at 37°C. with 0, 0.25, 1, 2.5 mg/mL of passion fruit skin extract and enoughsaline to achieve a final volume of 1 mL. Following incubation andcentrifugation, the supernatants were removed and the membrane wasresuspended in saline up to 1 mL. Thereafter, membrane ATPase activitieswere measured simultaneously in multi-well plates. The typical assaymixture contained RBC membrane (75 μg/mL), 18 mM histidine-imidazole (pH7.1, Sigma), 3 mM MgCI₂, 80 mM NaCI, 15 mM KCl, 0.2 mM CaCI₂, 0.1 mMEGTA, 0.1 mM Ouabain (Sigma) and 30 nM CaM (only for CaM-activated Ca²⁺pump, Sigma). After a 15 minute preincubation at 37° C., 5% SDS (Sigma)was added to the control groups. The enzymatic reaction was started with3 mM ATP. After 60 minutes at 37° C., the-reaction was stopped with 5%SDS; and the inorganic phosphate released was measured with an ammoniummolybdate/ascorbic acid mixture and absorption was measured at wavelength 820 nm by Microplate Autoreader (Bio-Tek Instruments, EL31 1.USA). For additional accuracy, a BCA assay was performed to determinethe final concentration of protein within the tubes at the end of eachassay. Membrane (25 μl) from the above experiment, 25 μl ddH₂O and 1 mLcolor reagent were added in a tube and then incubated for 30 mm at 37°C. Standard protein (Albumin, Sigma) at different concentrations wasincubated in the meantime. After incubation, each tube was cooled toroom temperature. Light absorbance was measured by spectrophotometer(Beckman Coulter, DU640) at X=562 nm. Protein concentration of themembrane was read from the standard curve. The activity of ATPase wascalculated by:

${{Activity}\mspace{14mu}{of}\mspace{14mu}{ATPase}} = \frac{{N\left( {P\; 1} \right)} \times 0.2778 \times {protein}\mspace{14mu}{concentration}}{{Initial}\mspace{14mu}{protein}\mspace{14mu}{concentration}\mspace{14mu}\left( {0.75\mspace{14mu}{mg}\text{/}{mL}} \right)}$

Example 4 Animals and Diets—SHR Studies

Spontaneously hypertensive rats (SHR), 6 weeks old, were kept at 22 to20° C. and 50% humidity during the experiment. 24 SHRs were divided into3 groups with 8 rats in each group. They were fed the following diets:basic diet, basic diet supplemented with the passion fruit extract at 50mg/kg, or basic diet supplemented with the passion fruit extract at 10mg/kg (Table 1). The amount of food intake, body weight and systolicblood pressure were recorded once a week. Systolic blood pressure wasmeasured by tail cuff method (Softron, Co. Ltd, Tokyo, Japan). After 8weeks of feeding, all the rats were sacrificed under anesthesia withNembutal (0.1 mg/100 g body weight, Wako, Co. Ltd., Japan). Thymus,spleen, liver and heart were isolated and weighed. No toxicity wasobserved.

Example 5 Nitric Oxide Measurement

Measurement of nitric oxide was carried out as previously described(Rockett, K A., Awburn, M M., Cowden, W B. & Clark, J A. (1991) Infect.Immun. 59:3280-3). Nitric oxide is easily converted to nitrite. Nitratewas measured for nitric oxide. Dilutions of NaNO₂ (BDH; Wako, Co. Ltd.,Japan) and test compounds were made in distilled water in 96-well,flat-bottom plates to a final volume of 50 ul. 20 microliters of NH₄Clborate buffer was added to all wells requiring analysis for nitricoxide/nitrite. 50 microliters of Griess reagent [1% sulfanilamide plus0.1% N-(1-napthyl) ethylenenediamine dihydrochloride (Wako, Co. Ltd.,Japan) in 2 M H₂SO₄] was then added to wells to be analyzed for nitricoxide/nitrite. The plate was read at 540 nm (test) and nitric oxide andnitrite concentrations were read directly from a nitrite standard curve.

Example 6 Human Study

People with hypertension, 14 men aged 57.0±14.48 y (mean±SD) and 16women aged 57.56±12.75 y (mean±SD), were included in the study. Patientshad hypertension of stage 1 or 2 according to the guidelines of theJoint National Committee on Detection, Evaluation, and Treatment of HighBlood Pressure. In repeated blood pressure measurements, they hadsystolic blood pressure (SBP) between 144 and 210 mm Hg and diastolicblood pressure (DBP) between 80 and 120 mm Hg. The exclusion criteriaincluded those with renal or cardiac disease, taking oralcontraceptives, use of tobacco and alcohol, or taking any vitaminsupplements other than a single, daily multivitamin tablet. All ofsubjects included were taking antihypertensive combination therapyincluding diuretic, beta-blocker and ACE-inhibitor. More than onehundred were screened to select the thirty participants, none of whichdropped out during the study.

At the beginning of the study, the passion fruit and placebo groups didnot differ in mean blood pressure (SBP, 176.60±4.90 vs. 179.67±3.79 mmHg and DBP, 103.27±2.30 vs. 104.33±2.06 mm Hg), age, sex, height,weight, heart rate, and pretreatment medication against hypertension orpattern of ECG.

The study was approved by Mashhad University's Human Subjects Committee.After providing informed consent and the one week withdrawal of anyprevious antihypertensive treatment except Triamterene-H in twosubjects, eligible patients entered a four week, double-blind, placebocontrolled, parallel group study. Patients were requested to attendclinic for follow-up every week during the study to assess side-effects.In addition, blood pressure and heart rate were measured. At the firstvisit, a complete medical history and a physical examination, includingelectrocardiogram, were carried out. Blood pressure was measured by aregistered nurse, after the subject had been sitting for 10 min rest.Korotkoff phases I and V were taken as the systolic and diastolic bloodpressures, respectively. Repeated readings were taken at 2 minuteintervals for a total of 3 sitting measurements at each visit. Averagesof repeated measurements at a given visit were recorded. At the secondvisit, blood pressure and heart rate were assessed again and patientswere randomized to receive twice daily dosing of 2 mg/lb/day (maximum400 mg/day) of a statistical formula of passion fruit pill or asimilarly appearing placebo for 4 weeks. The data for week 1 and week 0were combined as baseline values. In the last visit, for each patient anECG was performed and the study drug was collected. All changes inconcomitant medications and clinical adverse events, either volunteeredor elicited by questioning, at baseline and follow-up visits wererecorded, with none reported.

Compliance was evaluated by tablet counting. During the four-week periodof treatment, all the subjects took 100% percent of the pills providedin a blinded fashion. All tests were two to four hours after the lastconsumption of pills.

Example 7 Isolation of Edulilic Acid from Passion Fruit Peel Extract

Passion fruit peel extract prepared according to Example 1 was dissolvedin 50% aqueous ethanol and was treated on a Sephadex LH20 column andeluted with 50% aqueous ethanol. The chromatographic fractions werecollected in 20 ml tubes with the aid of a fraction collector. Fractionswere monitored by thin layer chromatography using cellulose TLCdeveloped with 6% aqueous acetic acid and visualized under UV. Underthis condition, the novel compound (Rf 0.8) co-eluted with the coloredanthocyanins fraction (Rf 0.4-0.5). This fraction was collected andconcentrated and re-chromatographed on a column of MCI GEL CHP 20Ppurchased from Mitsubishi Chemical Industries Ltd. Using 15% aqueousmethanol as the eluating solvent. Fractions were collected and monitoredby cellulose TLC developed with tertiary BuOH—AcOH—H₂O (3/1/1 v/v).Fractions containing the novel compound (Rf 0.9 with this solvent) werecollected and the solvent evaporated and the residue was freeze dried.

A high resolution electrospray ionization mass spectrum of edulilic acidwas made on a MARINER Biospectrometry Workstation at the VictoriaUniversity of Wellington, New Zealand operating on the negative ion modeto give (M-H)-¹ peak at 301.09691 which corresponded to the molecularformulae of C₁₃H₁₈O₈. Various NMR studies (1H, 13C, COSY, HMQC, HMBC andNOSEY) and mass spectrometry were conducted on the edulilic acid. Table1 contains data from the NMR studies.

TABLE 1 ¹H and ¹³C NMR Spectral Assignment for Edulilic acid Recorded inD₂O δ 13C δ1H (HMQC) HMBC C/H position 1 29.1 2.70(m) C1, C2, C3, C4,C5, C6 2 31.8 2.37(bs) C3, C4 3 130.1 6.84(d) C1, C2, C4, C5 4 150.56.52(d) C1, C2, C3, C5 5 156.3 — 6 132.7 — 7 167.6 — glucose C1′ 103.54.57(d) C2′, C3′, C6 C2′ 76.4 3.30(m) C2′, C2′, C3′ C3′ 75.9 3.35(m) C4′C4′ 73.7 3.32(m) C3′, C4′, C5′ 69.5 3.30(m) C3′, C4′, C6′ C6′ 60.63.53(dd), 3.67(dd) C5′ C7 (CO) 167.6 —

On this basis the chemical structure of edulilic acid is as given below.The beta-linkage of the glucose residue is assigned due the largeJ-coupling magnitude (J=7.3 Hz) of the anomeric proton.

The drawing of the molecule was prepared using ACD/ChemSketch Software,which provided the IUPAC name(2E)-cyclopent-2-en-1-ylidene((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)aceticacid. The compound has been identified throughout this specification as“edulilic acid”, as having originally been discovered in the fruit ofPassiflora edulis. The structure of edulilic acid depicted above is theE or trans isomer. Edulilic acid is expected to undergo cis/transenolization to the Z or cis form and back. The electron shift can gofrom the C-2 protons through the acetic acid moiety, or from the aceticacid moiety and travel in the opposite direction to afford the samecis/trans result.

The NOESY spectrum of edulilic acid in D₂O shows that there is someinteraction of the sugar anomeric proton with the methylene protons onC-1 as numbered in the structure shown earlier (FIGS. 6 a and 6 b). Thisis only possible if the carboxylic acid moiety is on the double bondside of the cyclopentene ring. Further studies using a Dreiding modelfor edulilic acid show that the orientation of the sugar moiety thatoffer the least crowding to the cyclopentene ring indeed placed theanomeric proton in close proximity to the methylene protons on C-1.

Example 8 Detection of Edulilic Acid in the Pulp of Passion Fruit

An aqueous extract of passion fruit pulp was prepared in a similarmanner as described in Example 1 with respect to the skin, and itschemical profile was examined using HPLC. A different HPLC solventprogramming using methanol instead of acetonitrile was used successfullyto resolve the edulilic acid and prunasin peaks. Also, instead of UVdetection Evaporative Light Scattering Detection (ELSD) was used forbetter detection of weak UV absorbing compounds such as prunasin. FIGS.7 a and 7 b show the HPLC chromatograms of the extracts of the skin andpulp respectively obtained under such conditions. While the skin andpulp extracts were clearly distinguishable by their HPLC profile, it wasalso apparent that both edulilic acid and prunasin were present in bothextracts.

The foregoing description and drawings comprise illustrative embodimentsof the present inventions. The foregoing embodiments and the methodsdescribed herein may vary based on the ability, experience, andpreference of those skilled in the art. Merely listing the steps of themethod in a certain order does not constitute any limitation on theorder of the steps of the method. The foregoing description and drawingsmerely explain and illustrate the invention, and the invention is notlimited thereto, except insofar as the claims are so limited. Thoseskilled in the art that have the disclosure before them will be able tomake modifications and variations therein without departing from thescope of the invention.

1. A method of preparing a passion fruit extract containing edulilicacid, comprising the steps of: (a) extracting the passion fruit withwater to provide an aqueous extract and a solid residue; (b) contactingthe aqueous extract with a non-ionic polymeric matrix resin to adsorb atleast edulilic acid from the aqueous extract; and (c) eluting the atleast edulilic acid from the matrix to obtain said passion fruitextract.
 2. A method as defined in claim 1, wherein the elution stepuses an organic solvent.
 3. A method as defined in claim 2, wherein theorganic solvent is selected from the group consisting of ethanol,isopropyl alcohol, methanol, 1-propanol, and acetone.
 4. A method asdefined in claim 1, further comprising the step of washing the polymericmatrix with water after contact with the aqueous extract and prior tothe elution step.
 5. A method according to claim 1, wherein only thepassion fruit skin is used.